Herpesviridae is a family of envelope DNA viruses comprising three subfamilies, alpha, beta and gamma herpesviridae. The alpha subfamily includes herpes simplex virus (HSV) 1 and 2, and varicella zoster virus (VZV). The beta subfamily includes cytomegalovirus (CMV) and human herpes virus 6 (HHV-6) and human herpes virus 7 (HHV-7). The gamma subfamily includes Epstein-Barr virus (EBV)and human herpes virus (HHV-8).
The human herpes viruses are responsible for a variety of disease states from sub-clinical infections to fatal disease states in the immunocompromised. As one example, VZV is known to cause a number of serious diseases: chickenpox, shingles and post-herpetic neuralgia [S. Straus, Ann. Neurol., 35:S11-S 12 (1994)]. As another example, HSV-1 is acquired in childhood when it causes a self-limiting gingivostomatitis. The virus remains latent in the dorsal root ganglia and is reactivated later in life as cold sores in about one third of the population. HSV-1 is also a cause of keratitis, resulting in more than 300,000 cases per year in the US. HSV-2 is usually acquired through sexual contact and gives rise to genital herpes. Human CMV is a ubiquitous opportunistic pathogen that can result in life threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer and transplant patients.
Each of these members of the herpes virus families encodes a serine protease that is essential for its replication [F. Liu, & B. Roizman, J. Virol, 65:5149-5156 (1991) (Liu I); F. Liu & B. Roizman, Proc. Natl. Acad. Sci. 89:2076-2080 (1992) (Liu II). F. Liu & B. Roiznan, J. Virol, 67:1300-1309 (1993) (Liu III); A. R. Welch et al., J. Virol, 67:7360-7372 (1993) (Welch I); E. Z. Baum et al., J. Virol, 67:497-506 (1993); J. T. Stevens et al., Eur. J Biochem., 226:361-367 (1994); A. R. Welch et al., J. Virol., 69:341-347 (1993) (Welch II); D. L. Hall & P. L. Darke, J. Biol. Chem., 270:22697-22700 (1995); Weinheimer et al., J. Virol, 67:5813-5822 (1993); M. Gao et al., J. Virol., 68:3702-3712 (1994); C. L. DiIanni et al., J. Biol. Chem., 268:25449-25454 (1993) (DiIanni I); C. L. DiIanni et al., J. Biol. Chem. 269:12672-12676 (1994) (DiIanni II); P. J. McCann III et al., J. Virol., 68:526-529 (1994)]. These proteases each provide a potential target for therapeutic intervention.
The proteases from these viruses are encoded as precursor proteins that catalyze their own cleavage to produce an N-terminal domain of approximately 28 kDa having full or increased catalytic activity. These protease domains show some degree of sequence homology--20% to 40% identity between members of different subfamilies and as high as 90% identity within each subfamily. They show little sequence homology to any other known protein, including the absence of the conserved G-X-S/C-G-G [SEQ ID NO: 12] for chymotrypsin-like and G-T-S-M/A [SEQ ID NO: 13] for subtilisin-like proteases. The known herpes virus proteases all cleave a peptide bond between an alanine and a serine, but their substrate specificity beyond the scissile bond are different [A. Welch et al., J. Virol. 69, 341-347 (1993)].
Each known serine protease has its characteristic set of functional amino acid residues arranged in a particular three dimensional configuration to form an active site. Knowledge of the active site of such proteases and their three dimensional structure permits the use of methods of structure-based drug design to identify, and develop inhibitors of the proteases [C. Verlinde and W. Hol, Structure, 2:577-587 (July 1994); 1. D. Kuntz, Science, 257:1078-1082 (August 1992)]. Because the proteolytic activity of the herpesvirus-encoded protease plays an essential role in virus capsid maturation, inhibitors of the protease would thus inhibit infectious virus particle formation and thereby exert an antiviral action. For serine proteases of which trypsin is a protype, the active site is formed by Ser, His and Asp [H. Neurath, Science, 224:350-357 (April 1984)]. These three residues are known as the catalytic triad.
There is a need in the art for novel protease active sites and catalytic sequences to enable identification and structure-based design of protease inhibitors, which are useful in the treatment or prophylaxis of viral diseases caused by viruses of the herpes family, as well as other diseases in which the target enzyme may share catalytic domains with those of the herpes family.